Antiviral compounds

ABSTRACT

The present invention relates to certain 5-substituted pyrimidine nucleosides and pharmaceutically acceptable derivatives thereof and their use in the treatment and prophylaxis of varicella zoster virus, cytomegalovirus and Epstein Barr virus infections. Also provided are pharmaceutical formulations and processes for the preparation of the compounds according to the invention.

This is a continuation of copending application Ser. No. 07/132,478 ,filed on Dec. 14, 1987, now abandoned.

The present invention relates to novel pyrimidine nucleosides and theiruse in medical therapy particularly for the treatment of herpes virusinfections.

Of the DNA viruses, the herpes group is the source of the most commonviral illnesses in man. The group consists of herpes simplex virus(HSV), varicella zoster (VZV), cytomegalovirus (GMV) and Epstein-Barrvirus (EBV).

Varicella zoster virus (VZV) is a herpesvirus which causes chicken-poxand shingles. Chicken-pox is the primary disease produced in a hostwithout immunity and in young children is usually a mild illnesscharacterised by a vesicular rash and fever. Shingles or zoster is therecurrent form of the disease which occurs in adults who were previouslyinfected with varicella-zoster virus. The clinical manifestions ofshingles are characterised by neuralgia and a vesicular skin rash thatis unilateral and dermatomal in distribution. Spread of inflammation maylead to paralysis or convulsions. Coma can occur if the meninges becomeaffected. In immunodeficient patients VZV may disseminate causingserious or even fatal illness. VZV is of serious concern in patientsreceiving immunosuppressive drugs for transplant purposes or fortreatment of malignant neoplasia and is a serious complication of AIDSpatients due to their impaired immune system.

In common with other herpes viruses, infection with CMV leads to alifelong association of virus and host and, following a primaryinfection, virus may be shed for a number of years. Clinical effectsrange from death and gross disease (microcephaly, hepatosplenomegaly,jaundice, mental retardation) through failure to thrive, susceptibilityto chest and ear infections to a lack of any obvious ill effect. CMVinfection in AIDS patients is a predominant cause of morbidity as; in80% of the adult population, it is present in a latent form and can bere-activated in immuno-compromised patients. Epstein-Barr virus (EBV)causes infectious mononucleosis, and is also suggested as the causativeagent of nasopharyngeal cancer, immunoblastic lymphoma, Burkitt'slymphoma and hairy leukoplakia.

Attention has focussed on nucleoside analogues for the treatment ofherpes viral infections. One compound, originally of interest as auseful intermediate, is 2'-deoxy-5-ethynyluridine, the synthesis ofwhich is disclosed by Barr et al. (J. Chem. Soc. Perkin Trans. I (1978),1263). This compound was tested for antiviral activity in vitro againstvaccinia and herpes simplex for example as described by Walker et al(Nucleic Acid Res., Special Pub. No. 4, 1978) and in U.S. Pat.Specification No. 4424211 but did not demonstrate any effect of use inhuman medicinal chemotherapy

U.S. Pat. No. 4,863,906 describes and claims the use of2'-deoxy-5-ethynyluridine and its pharmaceutically acceptablederivatives in the treatment of human virus infections caused bycytomegalovirus (CMV) or varicella zoster virus (VZV).

We have now surprisingly discovered that certain other novel pyrimidinenucleosides characterised by the presence of an unsaturated grouping inthe 5-position are of particular value in medical therapy particularlyfor the treatment of certain viral infections as described below. Thesecompounds also have the advantage that they have been found to possess arelatively low level of toxicity as determined by cell culture toxicityexperiments in vitro.

The novel pyrimidine nucleosides referred to above may be represented bythe following general formula (I): ##STR1## wherein X represents avinylene or ethynylene group, R¹ represents an oxo or imino group; R²represents hydrogen or a C₁₋₂ alkyl or a C3-4 branched or cycloalkylgroup e.g. isopropyl or cyclopropyl; R³ represents a hydrogen atom or anacyl e.g. C₁₋₄ alkanoyl or benzoyl group optionally substituted forexample by one or more halogen, alkyl, hydroxy or alkoxy substituents;and R⁴ represents a hydrogen atom or a hydroxy group provided that (a)when R¹ is imino, R³ is hydrogen and R⁴ is hydrogen or hydroxy, --X--R²does not represent a vinyl, ethynyl or propenyl group; (b) when R¹ isoxo; R³ is hydrogen and R⁴ is hydrogen or hydroxy, --X--R² does notrepresent a vinyl, ethynyl, 1-propenyl, 1-propynyl, 1-butenyl, 1-butynylor 3,3,3-trimethyl-1-propynyl group; or a pharmaceutically acceptablederivative thereof.

It will be appreciated that when R³ is not an acyl group, the compoundof formula (I) may exist in its tautomeric form.

The above-mentioned pyrimidine nucleosides also include thepharmaceutically acceptable derivatives of such compounds, ie. anypharmaceutically acceptable salt, ester, or salt of such ester, or anyother compound which, upon administration to a human subject, is capableof providing (directly or indirectly) the antivirally active metaboliteor residue thereof.

Such pyrimidine nucleosides and their derivatives will be hereinafterreferred to as the compounds according to the invention. Preferredcompounds of formula (I) include those wherein

(a) X represents an ethynylene group especially when R² represents ahydrogen atom or a methyl group.

(b) R³ represents a hydrogen atom or a benzoyl group; and/or

(c) R⁴ represents a hydrogen atom especially when X represents anethynyl group and R² represents a methyl group.

The following novel compounds are preferred compounds according to theinvention particularly by virtue of their especially potent antiviralactivity, particularly against VZV and in some cases CMV.

(a) 3-N-benzoyl-2'-deoxy-5-ethynyluridine

(b) 3-N-benzoyl-2'-deoxy-5-propynyluridine

(c) 2'-deoxy-5-(1-propynyl)cytidine

(d) 1-(β-D-arabinofuranozyl)-5-propynylcytosine

Particularly preferred compound are b) and c) which have shown potentanti-VZV activity.

Further novel compounds of the invention include:

(j) 1-(β-D-arabinofuranozyl)-3-N-benzoyl-5-propynyluracil

(k) 1-(β-D-arabinofuranozyl)-3-N-benzoyl-5-ethynyluracil

(l) 3-N-benzoyl-2'-deoxy-5-vinyluridine

(m) 1-(β-D-arabinofuranozyl)-3-N-benzoyl-5-vinyluracil

Also pharmaceutically acceptable salts and esters of such compounds Theabove compounds all have particularly high activity against VZV, whilecompound a) also has high activity against CMV.

The present invention further includes a method for the treatment of aviral infection selected from VZV, CMV and EBV infections whichcomprises treating a subject with an effective amount of a compoundaccording to the invention.

Examples of the clinical conditions caused by such herpes viruses asCMV, VZV and EBV infections which may be treated in accordance with theinvention include those referred to above.

Preferred mono- and di-esters according to the invention includecarboxylic acid esters in which the non-carbonyl moiety of the estergrouping is selected from straight or branched chain alkyl, alkoxyalkyl(e.g. methoxymethyl), carboxyalkyl (e.g. carboxyethyl), aralkyl (e.g.benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionallysubstituted by halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy); sulphonate esterssuch as alkyl, or aralkylsulphonyl (e.g. methanesulphonyl); and mono-,di- or tri-phosphate esters which may or may not be blocked, amino acidsesters and nitrate esters. With regard to the above-described esters,unless otherwise specified, any alkyl moieties present in such estersadvantageously contain 1 to 18 carbon atoms, particularly 1 to 4 carbonatoms. Any aryl moiety present in such esters advantageously comprises aphenyl group. Any reference to any of the above compounds also includesa reference to a pharmaceutically acceptable salt thereof.

Salts according to the invention which may be conveniently used intherapy include physiologically acceptable base salts, eg derived froman appropriate base, such as alkali metal (e.g. sodium), alkaline earthmetal (e.g. magnesium) salts, ammonium and NX⁺ ₄ (wherein X is C₁₋₄alkyl) salts.

The compounds according to the invention may be administered by anyroute appropriate to the condition to be treated, suitable routesincluding oral, rectal, nasal, topical (including buccal andsublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural). Itwill be appreciated that the preferred route may vary with, for example,the condition of the recipient.

For each of the above-indicated utilities and indications the amountrequired of the individual active ingredients will depend upon a numberof factors including the severity of the condition to be treated and theidentity of the recipient and will ultimately be at the discretion ofthe attendant physician. In general, however, for each of theseutilities and indications, a suitable, effective dose will be in therange 0.1 to 250 mg per kilogram body weight of recipient per day,preferably in the range 1 to 100 mg per kilogram body weight per day andmost preferably in the range 5 to 30 mg per kilogram body weight perday; an optimum dose is about 15 mg per kilogram body weight per day(unless otherwise indicated all weights of active ingredient arecalculated as the parent compound; for salts and esters thereof thefigures would be increased proportionately.) The desired dose may ifdesired be presented as two, three, four or more sub-doses administeredat appropriate intervals throughout the day. These sub-doses may beadministered in unit dosage forms, for example, containing 10 to 1000mg, preferably 20 to 500 mg and most preferably 100 to 400 mg of activeingredient per unit dosage form.

While it is possible for the compounds to be administered alone it ispreferable to present them as pharmaceutical formulations. Theformulations of the present invention comprise at least one activeingredient, as above defined, together with one or more acceptablecarriers thereof and optionally other therapeutic ingredients. Thecarrier(s) must be "acceptable" in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipients thereof.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural) administration. The formulations may conveniently be presentedin unit dosage form and may be prepared by any of the methods well knownin the art of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

For infections of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient in an amount of, for example,0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to10% w/w. When formulated in an ointment, the active ingredients may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w"w of a polyhydric alcohol, i.e. an alcoholhaving two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol sorbitol, glycerol and polyethylene glycol andmixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulphoxide and relatedanalogues.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While this phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifer(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulphate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus the cream should preferably be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for nasal administration wherein the carrier is asolid include a coarse powder having a particle size for example in therange 20 to 500 microns which is administered in the manner in whichsnuff is taken, i.e. by rapid inhalation through the nasal passage froma container of the powder held close up to the nose. Suitableformulations wherein the carrier is a liquid, for administration as forexample a nasal spray or as nasal drops, include aqueous or oilysolutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon- aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The compounds according to the invention may be prepared by any of themethods known in the art for the preparation of the same or similarcompounds e.g. see U.S. Pat. Specification No. 4424211, or Robins M. J.,and Barr, P. J., J. Org. Chem. (1983) 1854-1862, and in particular theprocesses described in the Examples given hereinafter.

The present invention also provides a process for the preparation of acompound according to the invention which comprises:

A. condensing a compound of formula ##STR2## (wherein R² is ashereinbefore defined, X represents an ethynylene group, R¹ _(a)represents a protected hydroxy or amino group and M¹ represents ahydroxy protecting group) with a compound of formula ##STR3## (wherein Yrepresents a halogen atom, M² and M³ each represents ahydroxy-protecting group and R⁴ _(a) represents hydrogen or a protectedhydroxy group);

B. reacting a compound of formula ##STR4## wherein R¹, M², M³ and R⁴_(a) are as defined above and Z is a leaving group, with a compoundcapable of providing the necessary grouping of formula --X--R² (in whichX is ethynylene and R² is as defined above) to form a compound offormula (I) in which X is ethynylene and R³ is hydrogen; and,

C. reacting a compound of formula ##STR5## (wherein Q is an appropriateleaving group and X, R², M¹, M² and R⁴ _(a) are as defined above) withan agent serving to replace the group Q with an amino group; to form acompound in which X is ethynylene and R³ is hydrogen; and

optionally thereafter or simultaneously therewith, performing either orboth of the following, in any desired order:

i) removing any remaining protecting groups;

ii) where the resulting compound is a compound of formula (I),converting it into a pharmaceutically acceptable derivative thereof or,where the resulting compound is pharmaceutically acceptable derivative,converting it into a different pharmaceutically acceptable derivative ora compound of formula (I).

With regard to process A), the starting materials may be protected withconventional protecting groups such as acyl groups, e.g. alkanoyl oraroyl groups such as p-toluoyl, or triakylsilyl groups such as thetrimethylsilyl group, the M¹ and R¹ _(a) protecting groups beinggenerally silyl protecting groups. The halogen group Y of the sugarresidue (formula (III)) is conveniently chlorine and the reactioncarried out in the presence of a Lewis acid catalyst, for examplestannic chloride, in a appropriate solvent, such as 1,2-dichloroethane.The parent compound can then be obtained, following anomeric separation,by treatment with alcoholic base, such as sodium methoxide in methanol.This process is also described by Barr, et al., in J. Chem. Soc., PerkinTrans 1 (1978), 1263 et seq.

The protecting groups can subsequently be removed by acid or basehydrolysis, acyl groups being advantageously removed by base hydrolysisand silyl groups by acid hydrolysis.

Regarding process B), this is exemplified by Robins, M. J., and Barr, P.J., in J. Org. Chem. (1983), 48, 1854 et seq. A 5-halogenated nucleosidesuch as 2'-deoxy-5-iodouridine in a suitably protected form, for examplewith the protecting groups referred to above, can be subjected to acatalysed coupling reaction, for example with a palladium catalyst, withan appropriate protected acetylene, such as trimethylsilylacetylene, inthe presence of an organic base, such as triethylamine, and anothermetal catalyst, for example a copper (I) salt, at an elevatedtemperature such as 50° C. to give the protected acetylenic nucleoside.A preferred palladium catalyst is bis(triphenylphosphine)palladiumdichloride and a preferred copper catalyst is cuprous iodide. The parentcompound can readily be obtained by removal of any protecting groups forexample by treatment with alcoholic base, such as sodium methoxide inmethanol.

In process C), the leaving group Q is advantageously a suitableheterocyclyl group advantageously a 1,2,4-triazol-1-yl group or a haloe.g. chloro group, the removal of the group Q being suitably effected bytreatment of the compound of formula (V) with ammonia.

The compound of formula (V) may be prepared for example by treating thecorresponding 4-oxo compound with an appropriate functionalising agentserving to introduce the leaving group Q for example by treatment with1,2,4-triazole, conveniently in the presence of a condensing agent suchas 4-chlorophenylphosphodichloridate, e.g. in a base solventadvantageously pyridine or by treatment of the 4-oxo compound withthionyl chloride in dimethylformamide.

The above-mentioned starting materials may be prepared in conventionalmanner from known compounds using techniques that are known in the artfor example as described in Nucleic Acid Chemistry: Improved NewSynthetic Procedures, Methods and Techniques. Ed. L. B. Townsend and R.S. Tipson, Wiley Interscience (1978) and Nucleoside Analogues:Chemistry, Biology and Medical Applications Ed. R. T. Walker, E deClercq and F. Eckstein, NATO Advanced Study Institute, Plenum Press(1979). Examples of methods for the preparation of the startingmaterials are described below.

In process A), the compound of formula (II) in which R² is hydrogen andX is ethynylene may be prepared, for example, by halogenating5-acetyluracil with a suitable halogenating agent, such as phosphorylchloride, in the presence of base to yield a compound of formula (VI)##STR6## wherein Y is halogen. The compound of formula (VI) may then befurther treated with an inorganic base, such as aqueous potassiumhydroxide, and then protected, to yield the compound of formula (II).

In process B), the compound of formula (IV) particularly wherein Zrepresents a halogen atom such as iodine and R⁴ _(a) represents aprotected hydroxy group can be prepared for example by methods analogousto those described by Schinazi et al, J. Med. Chem, 1979, 22 (20) 1273.

The compounds of formula (I) in which X represents a vinylene group maybe prepared from corresponding compounds in which X represents anethynylene group for example by catalytic hydrogenation using anappropriate catalyst for example a Lindlar catalyst poisoned withquinoline, eg in an alcoholic solvent such as methanol or ethanol. Othermethods for preparing these vinylene compounds are described for exampleby S. G. Rahim et al, Nucleic Acids Research 1982 10(17), 5285.

The compounds of formula (I) in which R³ represents an acyl group may beprepared from corresponding compounds in which R³ represents a hydrogenatom for example by selective protection of the hydroxy groups in thesugar residue, eg using trialkylsilyl groups, and subsequently acylatingthe protected compounds using for example the appropriate acid chlorideor anhydride, advantageously in the presence of a base such as pyridineor triethlyamine which may also serve as a solvent medium for thereaction. The resulting acyl compound of formula (I) may then beobtained by deprotection, eg by removal of the trialkylsilyl groups byfor example treatment with an acid such as acetic acid.

Esters according to the invention may be prepared in conventional mannere.g. by treatment of the parent compound of formula (I) or an esterthereof (optionally protected) with an appropriate esterifying ortransesterifying agent respectively, for example, by treatment of2'-deoxy-5-ethynyluridine with an appropriate acid halide (e.g.chloride) or anhydride in the presence of base, conveniently pyridine,which may also be used as the solvent, any remaining protecting groupsbeing thereafter removed.

Salts according to the invention may also be prepared in conventionalmanner for example by reaction of the parent compound with anappropriate base to form the corresponding base salt. Other derivativesaccording to the invention can also be prepared in conventional manner.

The following examples illustrate the present invention.

Example 1 3-N-Benzoyl-2'-deoxy-5-ethynyluridine

To a stirred suspension of 2'deoxy-5-ethynyluridine (J. Med. Chem.,26(5), 661-6, (1983)) (0.3 g, 1.19 mmoles) in dry acetonitrile (8 ml)and chlorotrimethylsilane (0.5 ml) was added, with ice cooling,triethylamine (0.85 ml, 6.1 mmoles) and the whole was stirred at roomtemperature for 2 hrs. Benzoylchloride (0.18 ml, 1.54 mmoles) was thenadded and the mixture stirred for a further 1.5 hrs and filtered. Thefiltrate was concentrated, the residue dissolved in ethanol (10 ml) andglacial acetic acid (0.4 g) was added. After stirring for 0.5 hr, thesolvents were evaporated and the residue purified by columnchromatography on silica gel eluting with methylene chloride/methanol(19:1) to give the title compound 0.2 g (47% yield) melting at 156°-7°C.

CHN Cal. C, 60.70,H, 4.49; N, 7.86%

Found C, 60.44; H, 4.336; N, 7.69%

Example 2 2'-Deoxy-5-propynylcytidine a)2'-deoxy-3',5'-di-O-acetyl-5-propynyluridine

0.8 ml of acetic anhydride was added to a solution of2'-deoxy-5-propynyluridine (J. Med. Chem., 26(5), 661-6, (1983)) (1 g,3.76 mmol) in 10 ml of dry pyridine. The mixture was stirred at roomtemperature 16 hrs., then evaporated to dryness to give a foamy solid.Recrystallisation from ethanol gave 0.98 g (75%) of the title compoundmelting at 148°-9° C.

CHN calculated C, 54.86; H, 5.143; N, 8.00%

Found C, 54.82; H, 5.114; N, 7.805%

b) 1-(2-Deoxy-3,5-di-O-acetyl-β-D-ribofuranosyl)-5-propynyl-4-(1,2,4-triazol-1-yl)pyrimidine-2(1H)-one

p-Chlorophenylphosphodichloridate (0.76 g, 3.10 mmol) was added to asolution of product of stage a) (0.7 g, 2 mmol) and 0.45 g of1,2,4-triazole (6.5 mmol) in 20 ml of dry pyridine. The solution wasstirred under dry N₂ at room temperature for 2 days then evaporated todryness. The resulting oil was eluted on a silica gel column with ethylacetate to give 0.33 g of starting material and 0.2 g (44% based on 0.4g of starting material) of the title compound, which decomposes furtheron standing at room temperature. This was not purified further butcarried onto the next step.

c) 2'-deoxy-5-propynylcytidine

Crude product of stage b) (0.2 g, 0.5 mmol) was dissolved in 5 ml ofDioxan/880NH₃ /H₂ O (3:2:1) and the mixture was left standing at roomtemperature for 16 hrs. It was then evaporated to dryness and theresidue co-evaporated with ethanol. The resulting white solid wasrecrystallised from ethanol to give 0.04 g (30%) of pure productdecomposing at 195°-200° C.

CHN calc. C, 54.34; H, 5.60; N, 15.85%

Found C, 54.19; H, 5.550; N, 15.33%

Example 3 1-(β-D-Arabinofuranozyl)-5-propynylcytosine a) O²,2'-Anhydrouridine

Uridine (10 g, 0.04 mole) was dissolved in 20 ml of warm, drydimethylformamide, and 11.4 g of diphenylcarbonate (0.06 m) and 0.2 g ofsodium bicarbonate were added. The solution was stirred and heated at150° C. until evolution of carbon dioxide ceased (30 min approx). Aftercooling the solution was poured into 200 ml of ether with rapidstirring. The resulting solid was filtered off, washed with ether, andrecrystallised from methanol to give 7.2 g (80%) of the title compound,as white crystals, melting at 235°-40° C.

b) 1 (β-D-Arabinofuranosyl)uracil

The product of Stage a) (7.0 g, 0.03 mole) was dissolved in 585 ml ofethanol/water (1:1) and 41 ml of 1M sodium hydroxide was added. Afterstirring at room temperature for 2.0 hr the solution was acidified to pH4-5 by portionwise addition of Dowex 50(H) ion exchange resin. The resinwas filtered off and washed with 100 ml of ethanol/water (1:1). Thefiltrate was evaporated to dryness, and the residue recrystallised fromethanol, to give 5.51 g (75%) of the title compound, as white crystals,melting at 220°-3° C.

c) 1 (B-D-Arabinofuranosyl)-5-iodouracil

The product of Stage b) (3.0 g, 12.3 mmole), 3.0 g of iodine (11.8mmole), 15 ml of chloroform, and 30 ml of 1M nitric acid were vigorouslystirred and refluxed together for 2.0 hr. After cooling, a crystallinesolid separated, which was filtered off, and washed thoroughly withether to remove excess iodine. The solid was recrystallised from waterto give 2.55 g (56%) of the title compound as white crystals melting at191°-3° C. (decomp).

d) 5-Iodo-1-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)uracil

Acetic anhydride (1.04 ml, 11 mmol) was added to a solution of 1 g ofproduct of stage (c) (2.7 mmol) in 10 ml of dry pyridine. After stirringfor 3 hours at room temperature, the solvent was evaporated and theresidue was co-evaporated with CH₂ Cl₂ several times. The CHN calc. C,54.34; H, 5.60; N, 15.85% residue was triturated with ethanol, the solidfiltered and dried to give 1.25 g (93%) of the title compound, meltingat 175°-9° C.

e) 5-Propynyl-1 (2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)uracil

A suspension of product of stage a) (1.16 g, 2.3 mmol), 35 mg of cuprousiodide and 35 mg of bis(triphenylphosphine)palladium (II) chloride in 95ml of dry triethylamine was stirred under dry N₂ for 15 mins. Propynegas was then bubbled through the mixture for 15 mins and the mixture wasstirred under an atmosphere of N₂ at 50° C. for 1 hr. The solution wasfiltered and the filtrate evaporated to dryness. The residue was takenup in CH₂ Cl₂ (30 ml) washed with 2×25 ml portions of 2% aqueousdisodium ethylenediamine tetracetic acid solution and 50 ml of water.The organic solution was dried (Na₂ SO₄) and evaporated andrecrystallisation of the residue from ethanol gave 0.38 g (40%) of thetitle compound melting at 150°-157° C.

CHN calc. C, 52.94; H, 4.902; N, 6.863%

Found C, 52.86; H, 4.827; N, 6.784%

f)5-Propynyl-1-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)-4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one

p-Chlorophenylphosphodichloridate (10.5 ml) was added to a solution ofproduct of stage b) (0.5 g, 1.2 mmol) and 0.4 g of 1,2,4-triazole in 10ml of dry pyridine. The solution was stirred at room temperature underan atmosphere of dry nitrogen for 6 days then evaporated to dryness. Theresidue was partitioned between dichloromethene (30 ml) and water (30ml). The organic layer was washed with 25 ml of water, dried (Na₂ SO₄)and evaporated, to give 0.65 g of crude product which was used in thenext stage of the synthesis.

g) 1-(β-D-arabinofuranosyl)-5-propynylcytosine

Crude product of stage c) (0.65 g, 1.39 mmol) was dissolved in 30 ml ofDioxan/88ONH₃ /H₂ O (3:2:1) and the solution was left standing at roomtemperature overnight. The solution was evaporated to dryness to give ayellow oil which was triturated with ethanol to give the pure product(0.22 g) melting at 241°-243° C. (deomposed).

CNH Calculated for C₁₂ H₁₅ N₃ O₅.0.7 H₂ O C, 49.05; H, 5.586; N, 14.30%

Found C, 49.28; H, 5.32; N, 13.95%

Example 4 3-N-Benzoyl-2'-deoxy-5 -propynyluridine

Dry triethylamine (1.1 ml) was added at O° C. to a stirred suspension of2'-deoxy-5-propynyluridine (J. Med. Chem., 26(5), 661-6 (1983)) (0.4 g,1.5 mmol) and 0.54 g of trimethylsilylchloride (4.97 mmol) in 10 ml ofdry acetonitrile. The mixture was stirred at room temperature for 2.5hours then 0.3 ml of benzoylchloride was added and stirring continuedfor a further 5 hours. A solid was filtered off, the filtrate wasevaporated to dryness, the residue was taken up in ethanol and 0.4 g ofacetic acid were added and the mixture was stirred at room temperaturefor 0.5 hours. The mixture was evaporated to dryness and the residue waseluted on a silica gel column with CH₂ Cl₂ /MeOH (9:1). Severalrecrystallisations from aqueous ethanol gave 0.14 g (25%) of theproduct, melting at 137°-140° C.

CHN calculated for C₁₉ H₁₈ N₂ O₆.0.2 H₂ O C, 60.96; H, 4.920; N, 7.489%

Found C, 60.69; H, 4.681; N, 7.478%

Example 5 2'-Deoxy-5-ethynyl-3-N-(p-toluoyl)uridine

To a stirred suspension of 2'-deoxy-5-ethynyluridine (J. Med. Chem.,26(5), 661-6, (1983)) (0.4 g, 1.6 mmol) in dry acetonitrile (15 ml) andchlorotrimethyl silane (0.7 ml, 5.6 mmol) was added, with ice cooling,triethylamine (1.13 ml, 8.1 mmol) and stirring was maintained at roomtemperature for 2.5 hr. Toluoylchloride (0.3 ml, 2.1 mmol) was thenadded and after stirring for a further 7 hr the mixture was filtered,the filtrate evaporated to dryness and the residue dissolved in ethanol(20 ml). Glacial acetic acid was then added and after stirring for 0.5hr at room temperature the solvent was evaporated and residual aceticacid coevaporated with ethanol. Purification of the product by thicklayer silica chromatography eluting with methanol/methylenechloride(1:24) followed by recrystallisation from methanol afforded titlecompound (0.03 g) melting at 176-9° C.

CHN calculated for C₁₉ H₁₈ N₂ O₆.0.2 EtOH C, 61.37; H, 5.08; N, 7.36%

Found C, 60.92; H, 4.87; N, 7.40%

The following examples illustrate pharmaceutical formulations accordingto the invention in which the active ingredient is a compound of formula(I).

    ______________________________________                                        Example A Tablet                                                              ______________________________________                                        Active ingredient      100    mg                                              Lactose                200    mg                                              Starch                 50     mg                                              Polyvinylpyrrolidone   5      mg                                              Magnesium stearate     4      mg                                                                     359    mg                                              ______________________________________                                    

Tablets are prepared from the foregoing ingredients by wet granulationfollowed by compression.

    ______________________________________                                        Ophthalmic Solution                                                           Example B                                                                     ______________________________________                                        Active ingredient        0.5                                                  Sodium chloride, analytical grade                                                                      0.9    g                                             Thiomersal               0.001  g                                             Purified water to        100    ml                                            pH adjusted to           7.5                                                  ______________________________________                                    

Example C: Tablet Formulation

The following formulations A and B are prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

    ______________________________________                                                         mg/tablet                                                                              mg/tablet                                           ______________________________________                                             Formulation A                                                            (a)  Active ingredient 250        250                                         (b)  Lactose B.P.      210         26                                         (c)  Povidone B.P.      15         9                                          (d)  Sodium Starch Glycollate                                                                         20         12                                         (e)  Magnesium Stearate                                                                               5          3                                                                 500        300                                              Formulation B                                                            (a)  Active ingredient 250        250                                         (b)  Lactose           150        --                                          (c)  Avicel PH 101      60         26                                         (d)  Povidone B.P.      15         9                                          (e)  Sodium Starch Glycollate                                                                         20         12                                         (f)  Magnesium Stearate                                                                               5          3                                                                 500        300                                              Formulation C                                                                 Active ingredient 100                                                         Lactose           200                                                         Starch             50                                                         Povidone           5                                                          Magnesium stearate                                                                               4                                                                            359                                                    ______________________________________                                    

The following formulations, D and E, are prepared by direct compressionof the admixed ingredients. The lactose used in formulation E is of thedirect compression type.

    ______________________________________                                                          mg/capsule                                                  ______________________________________                                        Formulation D                                                                 Active Ingredient   250                                                       Pregelatinised Starch NF15                                                                        150                                                                           400                                                       Formulation E                                                                 Active Ingredient   250                                                       Lactose             150                                                       Avicel              100                                                                           500                                                       ______________________________________                                    

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients(below) with a solution of povidone followed by the addition ofmagnesium stearate and compression.

    ______________________________________                                                            mg/tablet                                                 ______________________________________                                        (a)    Active Ingredient  500                                                 (b)    Hydroxypropylmethylcellulose                                                                     112                                                        (Methocel K4M Premium)                                                 (c)    Lactose B.P.        53                                                 (d)    Povidone B.P.C.     28                                                 (e)    Magnesium Stearate  7                                                                            700                                                 ______________________________________                                    

Drug release takes place over a period of about 6-8 hours and wascomplete after 12 hours.

Example D: Capsule Formulation Formulation A

A capsule formulation is prepared by admixing the ingredients ofFormulation D in Example C above and filling into a two-part hardgelatin capsule. Formulation B (infra) is prepared in a similar manner.

    ______________________________________                                                          mg/capsule                                                  ______________________________________                                               Formulation B                                                          (a)    Active ingredient                                                                              250                                                   (b)    Lactose B.P.     143                                                   (c)    Sodium Starch Glycollate                                                                        25                                                   (d)    Magnesium Stearate                                                                              2                                                                            420                                                          Formulation C                                                          (a)    Active ingredient                                                                              250                                                   (b)    Macrogol 4000 BP 350                                                                           600                                                   ______________________________________                                    

Capsules are prepared by melting the Macrogol 4000 BP, dispersing theactive ingredient in the melt and filling the melt into a two-part hardgelatin capsule.

    ______________________________________                                        Formulation D   mg/capsule                                                    ______________________________________                                        Active ingredient                                                                             250                                                           Lecithin        100                                                           Arachis Oil     100                                                                           450                                                           ______________________________________                                    

Capsules are prepared by dispersing the active ingredient int helecithin and arachis oil and filling the dispersion into soft, elasticgelatin capsules.

Formulation E (controlled Release Capsule)

The following controlled release capsule formulation is prepared byextruding ingredients a, b, and c using an extruder, followed byspheronisation of the extrudate and drying. The dried pellets are thencoated with release controlling membrane (d) and filled into a two-piecehard gelatin capsule.

    ______________________________________                                        Example E: Injectable Formulation                                                                       mg/                                                                           capsule                                             ______________________________________                                        (a)    Active Ingredient        250                                           (b)    Microcrystalline Cellulose                                                                             125                                           (c)    Lactose BP               125                                           (d)    Ethyl Cellulose           13                                                                           513                                                  Active ingredient        .0200  g                                             Sterile, pyrogen free phosphate buffer (pH 7.0) to                                                     10     ml                                     ______________________________________                                    

The active ingredient is dissolved in most of the phosphate buffer(35°-40° C.), then made up the volume and filtered through a sterilemicropore filter into a sterile 10 ml amber glass vial (type 1) andsealed with sterile closures and overseals.

    ______________________________________                                        Example F: Intramuscular injection                                            ______________________________________                                        Active Ingredient       0.20   g                                              Benzyl Alcohol          0.10   g                                              Glucofurol 75           1.45   g                                              Water for Injection q.s. to                                                                           3.00   ml                                             ______________________________________                                    

The active ingredient is dissolved in the glycofurol. The benzyl alcoholis then added and dissolved, and water added to 3 ml. The mixture isthen filtered through a sterile micropore filter and sealed in sterile 3ml glass vials (type 1).

    ______________________________________                                        Example G: Syrup Suspension                                                   ______________________________________                                        Active ingredient      0.2500  g                                              Sorbitol Solution      1.5000  g                                              Glycerol               2.0000  g                                              Dispersible Cellulose  0.0750  g                                              Sodium Benzoate        0.0050  g                                              Flavour, Peach 17.42.3169                                                                            0.0125  ml                                             Purified Water q.s. to 5.0000  ml                                             ______________________________________                                    

The sodium benzoate is dissolved in a portion of the purified water andthe sorbitol solution added. The active ingredient is added anddispersed. In the glycerol is dispersed the thickener (dispersiblecellulose). The two dispersions are mixed and made up to the requiredvolume with the purified water. Further thickening is achieved asrequired by extra shearing of the suspension.

    ______________________________________                                        Example H: Suppository                                                                               mg/suppository                                         ______________________________________                                        Active Ingredient (63 μm)*                                                                           250                                                 Hard Fat, BP (Witepsol H15 - Dynamit NoBel)                                                            1770                                                                          2020                                                 ______________________________________                                         *The active ingredient is used as a powder wherein at least 90% of the        particles are of 63 μm diameter or less.                              

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 μm sieve andadded to the molten base with mixing, using a silverson fitted with acutting head, until a smooth dispersion is achieved Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogenous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to 40° C. At a temperature of 38° C. to 40° C. 2.02 gof the mixture is filled into suitable plastic moulds. The suppositoriesare allowed to cool to room temperature.

    ______________________________________                                        Example I: Pessaries                                                                          mg/pessary                                                    ______________________________________                                        Active ingredient 63 μm                                                                      250                                                         Anhydrate Dextrose                                                                              380                                                         Potato Starch     363                                                         Magnesium Stearate                                                                               7                                                                            1000                                                        ______________________________________                                    

The above ingredients are mixed directly and pessaries prepared bydirect compression of the resulting mixture.

Antiviral and Toxicity Testing

Human cytomegalovirus (HCMV) is assayed in monolayers of either MRC5cells (human embryonic lung) or Detroit 532 cells (human foreskinfibroblasts) in multiwell trays. Activity of compounds is determined inthe plaque reduction assay, in which a cell monolayer is infected with asuspension of HCMV, and then overlaid with nutrient agarose in the formof a gel to ensure that there is no spread of virus throughout theculture. A range of concentrations of compound of known molarity wasincorporated in the nutrient agarose overlay. Plaque numbers of eachconcentration are expressed as percentage of the control and adose-response curve is drawn. From this curve the 50% inhibitoryconcentration (IC₅₀) is estimated.

Varicella zoster virus (VZV) is assayed in MRC5 cells by a similarmethod of that for HCMV with the omission of the agarose overlay.

An assay is performed in which virus-producing cells (P3HR-1) areexposed to drug for 14 days after which the EBV genome copies per cellare determined by EBV specific c-RNA-DNA hybridization. Epstein Barrvirus is assayed by the methods of Nonoyama & Pagano disclosed inNature: New Biology Vol. 233, pg. 103-4 1971. The IC₅₀ value given inthe results is the concentration required to inhibit the EBV genomeNo/cell by 50%

Cell toxicity is assessed in a cell growth inhibition assay.Subconfluent cultures of Vero cells grown on 96-well microtiter dishesare exposed to different dilutions of drug, and cell viabilitydetermined daily on replicate cultures using uptake of a tetrazolium dye(MTT). The concentration required for a 50% inhibition of cell viabilityat 96 hours is termed CCID₅₀.

The results are shown in the following Table.

                  TABLE                                                           ______________________________________                                                 IC.sub.50 (μM)                                                                          IC.sub.50 (μM)                                                                       CCID.sub.50 (μM)                           Example  VZV          HCMV      at 96 hr                                      ______________________________________                                        2        3.3          >20       >300                                          4        2.2          28.5      >500                                          ______________________________________                                    

We claim:
 1. A compound of formula (I)B ##STR7## wherein R¹ representsan oxo or imino group; R² represents hydrogen, a C₁₋₂ alkyl or a C₃₋₄branched or cycloalkyl group; R³ represents a hydrogen atom, or C₁₋₄alkanoyl or a benzoyl group optionally substituted by one or morehalogen, alkyl, hydroxy or alkoxysubstitutents; and R⁴ represents ahydrogen atom or a hydroxy group provided that (a) when R¹ is imino; R³is hydrogen and R⁴ is hydrogen or hydroxy; R² does not represent ahydrogen atom or methyl group; (b) when R¹ is oxo; R³ is hydrogen and R⁴is hydrogen or hydroxyl; R² does not represent a hydrogen atom, methyl,ethyl or tertbutyl group; or a pharmaceutically acceptable salt, esteror salt of such ester.
 2. A compound according to claim 1 wherein R¹ isoxo.
 3. A compound according to claim 1 or 2 wherein R⁴ is hydrogen. 4.A compound according to claim 1 wherein R³ is benzoyl.
 5. The compound3-N-Benzoyl-2'-deoxy-5-ethynyluridine.
 6. The compound2'-deoxy-5-(1-propynyl)cytidine.
 7. The compound1-(β-D-Arabinofuranozyl)-5-propynylcytosine.
 8. The compound3-N-Benzoyl-2'-deoxy-5-propynyluridine.
 9. A compound according to claim1 wherein the compound is1-(β-D-Arabinofuranozyl)-3-N-benzoyl-5-ethynyluracil.
 10. A compoundaccording to claim 1, wherein the compound is1-(β-D-arabinofuranozyl)-3N-5-propynyl uracil.
 11. A pharmaceuticalformulation suitable for use in combatting human VZV, CMV or EBVinfections comprising as active ingredient, an effective VZV, CMV or EBVtreatment amount of a compound according to claim 1 and apharmaceutically acceptable carrier therefor.
 12. A formulationaccording to claim 11 in a form for administration by injection.
 13. Aformulation according to claim 11 in a form of a sterile solution.
 14. Aformulation according to claim 11 in a form for topical administration.15. A formulation according to claim 14 in the form of a topicalointment or cream.
 16. A formulation according to claim 11 in a form fororal administration.
 17. A formulation according to claim 16 in the formof a tablet or capsule.
 18. A pharmaceutically acceptable salt, ester orsalt of such ester of the compound3-N-Benzoyl-2'-deoxy-5-propynyluridine.
 19. A pharmaceuticallyacceptable salt, ester or salt of such ester of the compound2'-deoxy-5-(1-propynyl)cytidine.
 20. A pharmaceutically acceptable salt,ester or salt of such ester of the compound3-N-Benzoyl-2'-deoxy-5-ethynyluridine.
 21. A method of treating VZM, CMVor EBV infection in a human comprising the administration to said humanof an effective VZV, CMV or EBV infection treatment amount of a compoundof formula I(B): ##STR8## wherein R¹ represents an oxo or imino group;R² represents hydrogen, a C₁₋₂ alkyl or a C₃₋₄ branched or cycloalkylgroup; R³ represents a hydrogen atom, or C₁₋₄ alkanoyl or a benzoylgroup optionally substituted by one or more halogen, alkyl, hydroxy oralkoxy substituents; and R⁴ represents a hydrogen atom or a hydroxygroup provided that (a) when R¹ is imino; R³ is hydrogen and R⁴ ishydrogen or hydroxy, R² does not represent a hydrogen atom or methylgroup; (b) when R¹ is oxo; R³ is hydrogen and R⁴ is hydrogen or hydroxy;R² does not represent a hydrogen atom, methyl, ethyl or tertbutyl group;or a pharmaceutically acceptable salt, ester or salt of such ester. 22.A method according to claim 21 wherein said infection is a CMVinfection.
 23. A method according to claim 21 wherein said infection isa VZV infection.
 24. A method according to claim 21 wherein saidinfection is a EBV infection.